The long-term objectives are presented in this proposal are: (1) To gain a better understanding of how cells ensure the correct folding of newly synthesized glycoproteins. (2) To determine how cells dispose of incorrectly folded glycoproteins. These objectives will be accomplished by studying two enzymes, protein disulfide isomerase (PDI) and peptide-N-glycanase (PNGase). In Specific Aim 1 we will relate the structure of yeast PDI, which we have recently determined in collaboration with Dr. Hermann Schindelin to the function of this enzyme in catalyzing the folding and generation of disulfide bonds in nascent proteins. In addition, we will solve the crystal structure of human PDI in order to determine the structural basis for its differences from yeast PDI. We have demonstrated that the other enzyme, PNGase, which deglycosylates misfolded proteins is part of a degradation complex that consists of five proteins that interact in vitro, and we have also collaborated with Dr. Hermann Schindelin in the structural analyses of various domains of PNGase including cocrystal structures with some of its binding partners. Specific Aim 2 is to investigate whether this multi-protein complex exists in vivo and if it functions in routing misfolded glycoproteins out of the lumen of the ER into the cytosol, where they are deglycosylated and degraded. These studies are of high medical relevance because there are numerous human disorders that involve misfolded proteins. Inefficient removal of misfolded (glyco) proteins results in aggregate formation, which occurs in Alzheimer's, Parkinson's, Huntington's, Creutzfeldt-Jakob, and Gaucher's disease, and many other degenerative disorders. An increasing number of human genetic diseases turn out to be caused by secretory protein trafficking problems including cystic fibrosis caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), emphysema and cirrhosis due to ?1-antitrypsin deficiency, and hemophilia in patients with defects in coagulation factors.